The unifying goal is to elucidate the catalytic mechanism of carbonic anhydrase by kinetic studies on three isozymes of this enzyme and some carefully selected site-specific mutants. A concurrent goal is to understand the properties and role of zinc-bound water in carbonic anhydrase and the kinetics of the proton transfer which converts it to zinc-bound hydroxide. These mechanistic studies will be based on 18-O exchange between CO2 and water and positional isotope exchange during catalysis. The exchanges will be measured using membrane-inlet mass spectrometry and analyzed to determine rates of intermediate steps in the catalysis. Additional information on intermediates will be obtained by studies of the visible absorption spectrum of Co(II)-substituted carbonic anhydrase during the progress curve for CO2 hydration. All of these data will be combined to give a quantitative model of catalysis. Site-specific mutants of carbonic anhydrase will be prepared to test the hypothesis that His 64 in carbonic anhydrase II enhances catalytic rate by shuttling protons from zinc-bound water to buffer in solution, and that Lys 64 in carbonic anhydrase III through electrostatic interactions decreases the catalytic rate of CO2 hydration and lowers the pKa of zinc-bound water to a value below 5. The hydrogen/deuterium fractionation factor of water as a ligand of metals is important in interpreting isotope effects and information on this property of water is lacking. This fractionation factor will be measured using a method developed in this lab and based on NMR relaxation times of the protons of water in the presence of paramagnetic metal ions. Values of the fractionation factor will be determined for simple inorganic complexes as well as for Co(II)-substituted carbonic anhydrase and other proteins. These studies will help to characterize water as a ligand of metals in metalloproteins and to interpret H/D solvent isotope effects in enzymatic reactions in which meta-bound water plays a role.